Low density lipoproteins (LDLs) play a major role in cholesterol transport in the blood and is a risk factor for arteriosclerosis. It is known that a small particle low density lipoprotein (hereinafter “small particle LDL”), which is especially smaller in particle size among LDLs and higher in density than standard LDL, is associated with a several fold increase in risk for arteriosclerosis as compared to normal LDL. Increase of small particle LDL is one of the major risk factors for arteriosclerosis. It is clinically very important to have a fractional measurement for small particle LDL.
The conventional methods for measuring small particle LDL use ultracentrifugation, electrophoresis, high speed liquid chromatography and the like. The ultracentrifugation method isolates small particle LDL by using differences in the density, and the quantities of cholesterol and protein therein are measured. Small particle LDL is fractionated in densities between 1.040 and 1.063 (Atherosclerosis, 48 p. 33-49, 1993: Atherosclerosis, 106, p. 241-253, 1994, etc.). However, this method requires expensive facilities, and it is time consuming to make measurements. The electrophoresis method measures the mobility and the particle diameter of a LDL using a polyacrylamide gel. The particle size of a small particle LDL is below 25.5 nm (JAMA, 260, p. 1917-21, 1988, etc.), and the relative mobility of LDLs (moving distance from VLDL to LDL divided by the moving distance from VLDL to HDL) is not less than 0.4 (Domyakukoka (arteriosclerosis), 25, p. 67-70, 1997). However, these methods are for measuring the degree of a small LDL particle in LDLs, and they are not used to obtain a quantitative measurement. Also, the number of samples that can be tested at one time is limited, and it takes a long time to make measurements. Recently, a method for measuring lipoprotein was invented. In this method, after electrophoresis, an agarose gel is stained for a lipid, and the staining pattern is analyzed using a computer, and a quantitative measurement of lipoprotein is obtained (Japanese Patent Publication Laid Open No. 2000-356641). This is a method for analyzing denatured LDL such as oxidized LDL, acetylated LDL, glycosylated LDL, MDA-LDL and the like. Small particle LDLs can not be measured accurately by this method. Since the analysis requires very expensive equipment, this is not suitable for general use.
Conventionally, in the measurement of a HDL, it is known that a combination of a polyanion with a divalent cation can be used as a separation agent to isolate the HDL by coagulating lipoproteins other than HDL. For example, methods using dextran sulfate-Mg2+ (Clin. Chem., 28, p. 1379-88, 1982, and the like), heparin-Mn2+ (J Lipid Res. 19, p. 65-76, 1978, and the like), heparin-Ca2+ (Arch. Biochem. Biophys., 170, p. 334-40, 1975, and the like)and phosphotungstic acid-Mg2+ (Clin. Chem., 23, p. 882-84, 1977, and the like)and the like are known. Further, a method has been reported for calculating the fractions of LDL and VLDL by stepwise precipitations of lipoproteins using several separation agents (Japanese Patent Publication Laid Open No. 7-294532, Rinsho-Byori, Special Edition 21, 82, 1975, and the like). Still further, a method for separating HDL using polyethylene glycol has also been reported (Ann. Clin. Biochem. 18 p. 177-81, 1981).
Furthermore, there have been conventional methods such as a method in which by stepwise precipitation of the lipoproteins using a plurality of separation agents, each lipoprotein is measured based on differences in their turbidity (Rinsho-Byori, Special Edition 21, 82, 1975, and the like), and a method in which a small particle LDL is suspended or dissolved according to differences in ionic strength and the small particle LDL is measured by differences in absorbency (Japanese Patent Publication Laid Open No. 2003-28882). However, because light absorbency is measured, specificity and accuracy have been insufficient.    Patent document 1 Japanese Patent Publication Laid Open No. 2000-356641    Patent document 2 Japanese Patent Publication Laid Open No. 7-294532    Patent document 3 Japanese Patent Publication Laid Open No. 2003-28882    Non-patent document 1 Atherosclerosis, 48 p. 33-49, 1993    Non-patent document 2 Atherosclerosis, 106, p. 241-253, 1994    Non-patent document 3 JAMA, 260, p. 1917-21, 1988    Non-patent document 4 Domyakukoka, 25, p. 67-70, 1997    Non-patent document 5 Clin. Chem., 28, p. 1379-88, 1982    Non-patent document 6 J Lipid Res. 19, p. 65-76, 1978    Non-patent document 7 Arch. Biochem. Biophys., 170, p. 334-40, 1975    Non-patent document 8 Clin. Chem., 23, p. 882-84, 1977    Non-patent document 9 Rinsho-Byori, Special Edition 21, 82, 1975    Non-patent document 10 Ann. Clin. Biochem. 18 p. 177-81, 1981